Project Summary Enteric infections have a staggering impact on global health and account for 15% of childhood mortality. The Gram-negative bacterium Salmonella enterica Typhimurium is among the most common sources of enteric infections, and for this reason, has been investigated extensively as a model intracellular pathogen. These studies have shown that Salmonella can reside within host cells in a specialized niche called the Salmonella Containing Vacuole (SCV), which provides protection from cytosolic immune sensors while undergoing bacterial replication. Understanding host pathways involved in the maintenance of the SCV is critical to designing novel therapeutic strategies that target this pathogen. When Salmonella in damaged SCV is recognized by cytosolic immune sensors, the membrane trafficking pathway of autophagy has been proposed to sequester the bacteria and target them for degradation in the lysosome. Support for this model has mainly been provided through in vitro studies. To determine whether autophagy has a similar role in vivo, we infected mice deficient in the essential autophagy protein Atg16L1. Surprisingly, we found that Atg16L1 mutant mice are protected against disease caused by Salmonella infection, which was associated with a striking reduction in intracellular bacterial burden. Thus, in addition to the well-established role of autophagy in degrading internalized bacteria, the autophagy pathway appears to have a major unknown role in enhancing Salmonella infection. This proposal aims to define how the autophagy pathway facilitates intracellular Salmonella replication in vivo. We will answer this question by defining the role of Atg16L1 in promoting intracellular Salmonella replication in vivo and by determining the relationship between autophagy and cytosolic sensors during in vivo Salmonella infection. Our findings challenge the current dogma, which is that the dominant role of autophagy during Salmonella infection is reduction of bacterial burden. This proposal will give new insight into how enteric pathogens have evolved to utilize essential cellular pathways for infection and may divulge unique cellular targets that can be manipulated to resolve a clinically relevant pathogen.